CN113024415B - 氟氯氰菊酯半抗原、人工抗原和抗体及其制备方法和应用 - Google Patents
氟氯氰菊酯半抗原、人工抗原和抗体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了氟氯氰菊酯半抗原、人工抗原和抗体及其制备方法和应用,本发明提供的氟氯氰菊酯半抗原既最大程度保留了氟氯氰菊酯的特征结构,使得氟氯氰菊酯半抗原的免疫原性明显增强,又具有可以与载体蛋白发生偶联的羧基;用氟氯氰菊酯半抗原与载体蛋白偶联后得到的氟氯氰菊酯人工抗原去免疫动物,更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体,经检测氟氯氰菊酯抗体的灵敏度可达0.1μg/L,与其他菊酯类杀虫剂的交叉反应率低,为后续建立氟氯氰菊酯的各种免疫分析方法提供了基础。
Description
技术领域
本发明属于食品安全检测领域。更具体地,本发明涉及氟氯氰菊酯半抗原、人工抗原和抗体及其制备方法和应用。
背景技术
氟氯氰菊酯(Cyfluthrin)是一种杀虫活性较高的拟除虫菊酯类杀虫剂,其对害虫的作用方式以触杀、胃毒作用为主,对多种鳞翅目害虫和某些地下害虫有良好的防效,被广泛应用于果蔬、茶叶和烟草中。但由于长期过量使用,其污染、残留所造成的环境问题已受到广泛关注。我国国家标准GB 2763规定了蔬菜及部分水果中氟氯氰菊酯的最大残留限量,因此建立相应农产品中氟氯氰菊酯的检测方法势在必行。
目前,国内外检测氟氯氰菊酯主要采用气相色谱法、气相色谱-质谱法和液相色谱串联质谱法等分析方法,存在样品前处理繁琐、检测时间长、仪器贵重等缺点,所以在我国无法得到广泛应用,并且不符合现场检测“在短时间内低成本对大量样品进行准确检测和筛选”的要求。而免疫学检测分析技术以其高灵敏、特异性高、快速、操作简便等优点在药物残留检测领域已被广泛应用,比起仪器等检验方法有很多优势。所以免疫分析为氟氯氰菊酯残留研究提供了一条新的分析检测方法。
在建立免疫学检测方法并应用该检测方法检测氟氯氰菊酯残留量时,关键技术在于能够获取到特异性强、灵敏度高的抗体,而要实现这一目标,前提条件就是得合成、制备出合适的氟氯氰菊酯半抗原。
发明内容
针对现有技术中存在的不足之处,本发明提供一种能最大程度保留氟氯氰菊酯的特征结构,又具有一定长度连接臂的半抗原以及这种半抗原的制备方法;以此半抗原制备的人工抗原、检测灵敏度高和特异性强的抗体;以及此半抗原的应用。
为了实现本发明的目的,第一方面,本发明提供一种氟氯氰菊酯半抗原,其具有如下结构式:
本发明提供的氟氯氰菊酯半抗原在氟氯氰菊酯的分子结构上引入羧基活性基团,从而可以与载体蛋白进行偶联得到人工抗原用于免疫;该氟氯氰菊酯半抗原保留了氟氯氰菊酯的所有特征基团,最小改变氟氯氰菊酯原有结构特征,同时与载体蛋白偶联后,突出的是氟氯氰菊酯本身独有的结构,为后续刺激动物免疫应答产生特异性更强、灵敏度更高的抗体奠定基础。
第二方面,本发明提供上述氟氯氰菊酯半抗原的制备方法,其包括如下步骤:
1)使羟基二氯菊酸与二氯亚砜反应形成酰氯,再与(alpha S)-4-氟-alpha-羟基-3-苯氧基-苯乙腈进行缩合反应,得到中间体1,所述中间体1具有结构式优选地,将羟基二氯菊酸溶解在吡啶中,加入二氯亚砜,室温搅拌反应2h后蒸干,再加入二氯甲烷溶解,加(alpha S)-4-氟-alpha-羟基-3-苯氧基-苯乙腈和三乙胺,室温搅拌反应3h,停止反应;优选地,羟基二氯菊酸、二氯亚砜、(alpha S)-4-氟-alpha-羟基-3-苯氧基-苯乙腈的摩尔比为1:1:1;
2)使中间体1与琥珀酸酐发生酯化反应,得到氟氯氰菊酯半抗原;优选地,中间体1、琥珀酸酐的摩尔比为1:1。
本发明根据氟氯氰菊酯的结构特点,通过全合成的方式,即羟基二氯菊酸与二氯亚砜形成酰氯,再与(alphaS)-4-氟-alpha-羟基-3-苯氧基-苯乙腈进行缩合成酯,使得在氟氯氰菊酯的三元环上引入羟基,进而再与琥珀酸酐进行酯化反应,从而在三元环上引入羧基间隔臂,使用的原料易得,反应操作较为简单,反应条件易于控制,制备的氟氯氰菊酯半抗原的纯度和收率较高。
第三方面,本发明提供一种氟氯氰菊酯人工抗原,其是载体蛋白和上述氟氯氰菊酯半抗原偶联得到的偶联物。所述氟氯氰菊酯人工抗原可以作为免疫原,也可以作为包被原。
进一步地,所述载体蛋白为牛血清白蛋白、鸡卵清白蛋白、人血清白蛋白或血蓝蛋白;优选牛血清白蛋白、鸡卵清白蛋白。
更具体的如:氟氯氰菊酯半抗原-牛血清白蛋白(BSA)形成的免疫原;氟氯氰菊酯半抗原-鸡卵清白蛋白(OVA)形成的包被原。
氟氯氰菊酯半抗原分子仅具有免疫反应性,而不具有免疫原性。因此,为了赋予氟氯氰菊酯半抗原分子以免疫原性,还需要将该氟氯氰菊酯半抗原分子与合适的载体蛋白分子偶联、结合在一起,由此产生既具有免疫反应性又具有免疫原性的氟氯氰菊酯人工抗原。
第四方面,本发明提供一种氟氯氰菊酯抗体,它是由上述氟氯氰菊酯人工抗原经动物免疫得到,其能与氟氯氰菊酯发生特异性免疫反应。
进一步地,所述氟氯氰菊酯抗体为单克隆抗体或多克隆抗体。另外,对于所述氟氯氰菊酯抗体,可以采用本领域常规方法来进行制备。
在一个具体的实施方案中,所述氟氯氰菊酯抗体为特异性针对上述氟氯氰菊酯半抗原的氟氯氰菊酯人工抗原的鼠源单克隆抗体。
采用本发明的氟氯氰菊酯人工抗原得到的氟氯氰菊酯抗体的效价、特异性、亲和力都较好,与其他菊酯类杀虫剂的交叉反应率低。
第五方面,本发明提供上述氟氯氰菊酯抗体在检测氟氯氰菊酯残留中的应用。
本发明通过氟氯氰菊酯人工抗原诱导免疫动物产生抗体,从而用于氟氯氰菊酯免疫检测分析中。
所述的氟氯氰菊酯免疫检测包括但不限于氟氯氰菊酯ELISA试剂盒、氟氯氰菊酯胶体金试纸条、氟氯氰菊酯时间分辨荧光试纸条。
借由上述技术方案,本发明至少具有如下优点及有益效果:
本发明提供的氟氯氰菊酯半抗原既最大程度保留了氟氯氰菊酯的特征结构,使得氟氯氰菊酯半抗原的免疫原性明显增强,又具有可以与载体蛋白发生偶联的羧基;用氟氯氰菊酯半抗原与载体蛋白偶联后得到的氟氯氰菊酯人工抗原去免疫动物,更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体,为后续建立氟氯氰菊酯的各种免疫分析方法提供基础。
本发明中氟氯氰菊酯半抗原的制备方法,使用的原料易得,反应操作较为简单,反应条件易于控制,制备的氟氯氰菊酯半抗原的纯度和收率较高。
采用本发明的氟氯氰菊酯人工抗原得到的氟氯氰菊酯抗体的效价、特异性、亲和力都较好,灵敏度可达到0.1μg/L,与其他菊酯类杀虫剂的交叉反应率低。
附图说明
图1是本发明氟氯氰菊酯半抗原的合成路线
具体实施方式
下面结合具体实施例进一步详细说明本发明,但实施例仅是本发明的优选实施方式,并不是对本发明的限定。
实施例1
一种氟氯氰菊酯半抗原的制备方法,其包括如下步骤:
1)取羟基二氯菊酸2.5g,加吡啶50mL溶解,加二氯亚砜1.53g,室温搅拌2h,直接蒸干,得到红色油状物;加二氯甲烷100mL溶解,加(alpha S)-4-氟-alpha-羟基-3-苯氧基-苯乙腈2.4g,加三乙胺2mL,室温搅拌3h,停止反应,加水80mL震荡,静置,分去水相,有机相无水硫酸钠干燥,蒸干,上硅胶柱,用石油醚和乙酸乙酯体积比为3:1的混合溶液洗脱分离,得到中间体1 1.25g;
2)取中间体1 1.25g,加乙腈100mL溶解,加三乙胺3mL充分搅拌,加琥珀酸酐0.55g,加热回流反应4h,停止反应,旋蒸,除去乙腈和三乙胺,得到粗品,加水100mL,用1mol/L HCl调节pH值到6,加乙酸乙酯70mL×3,萃取三次,合并有机相,蒸干,上硅胶柱,用二氯甲烷和甲醇体积比为10:1的混合溶液洗脱分离,得到氟氯氰菊酯半抗原。
实施例2
一种氟氯氰菊酯人工抗原的制备方法,步骤如下:
取实施例1制备的氟氯氰菊酯半抗原21mg,加N,N-二甲基甲酰胺(DMF)1mL溶解,加三乙胺20μL、氯甲酸异丁酯133μL,冷却至0~5℃,反应3h,得到半抗原溶液A液;取牛血清白蛋白(BSA)50mg,加0.05mol/L CB缓冲液溶解,得到B液;将A液滴加到B液中,4℃反应8h,用0.02mol/L PBS透析纯化3天,每天换液3次,得到与牛血清白蛋白偶联的氟氯氰菊酯人工抗原,分装,-20℃保存。
实施例3
一种氟氯氰菊酯人工抗原的制备方法,步骤如下:
取实施例1制备的氟氯氰菊酯半抗原12mg,加二甲基亚砜(DMSO)1mL溶解,加N-羟基丁二酰亚胺(NHS)10mg、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)12mg,室温反应3h,得到半抗原溶液A液;取鸡卵清白蛋白(OVA)50mg,加0.05mol/L PB缓冲液溶解,得到B液;将A液滴加到B液中,4℃反应8h,用0.02mol/L PBS透析纯化3天,每天换液3次,得到与鸡卵清白蛋白偶联的氟氯氰菊酯人工抗原,分装,-20℃保存。
实施例4
一种氟氯氰菊酯抗体,其制备方法为:
1.动物免疫
取健康的6~8周雌性Balb/c小鼠10只(分为A与B两组,每组5只),初次免疫用弗氏完全佐剂乳化后颈背部皮下多点注射,每只小鼠免疫剂量为200μg与牛血清白蛋白偶联的氟氯氰菊酯人工抗原;之后加强免疫每两周颈背部皮下多点注射一次,乳化用弗氏不完全佐剂;最后一次免疫使用生理盐水代替弗氏不完全佐剂,采用腹腔注射,注射剂量和前面几次相同。具体免疫步骤见表1。
表1小鼠免疫程序
第三次、四次、加强免疫后7d,对小鼠断尾取血,ELISA方法测定小鼠血清效价,具体步骤如下:
(1)用0.05mol/L pH9.6的碳酸盐缓冲液将与鸡卵清白蛋白偶联的氟氯氰菊酯人工抗原做1:1000稀释,每孔100μL包被酶标板,37℃孵育2h,甩掉包被液,以PBST洗涤1次,拍干;
(2)每孔加入150μL封闭液,37℃反应2h后倾去封闭液,拍干;
(3)每孔加入50μL以PBS倍比稀释的抗血清,25℃反应30min后倾去反应液,以PBST洗涤3~5次,每次间隔30s,拍干;
(4)加PBS稀释的辣根过氧化物酶标记的羊抗鼠抗抗体(1:1000)100μL/孔,25℃反应30min,以PBST洗涤3~5次,每次间隔30s,拍干;
(5)每孔加入底物显色液A液和B液各50μL,25℃避光反应15min,每孔加入50μL2mol/L的H2SO4溶液终止反应;
(6)酶标仪测定波长在450nm的OD值,以样品孔OD450接近于1的稀释倍数作为阳性血清的效价。
2.细胞融合
(1)饲养细胞制备:断颈处死8~10周龄Balb/c小鼠,浸泡在75%酒精中5min,随即放入超净工作台内,腹部朝上放于平皿内或固定于解剖板上。用眼科镊子夹起小鼠腹部皮肤,用剪刀剪一小口,注意切勿剪破腹膜,以免腹腔液外流和污染。然后用剪刀向上下两侧做钝性分离,充分暴露腹膜。用酒精棉球擦拭腹膜消毒。用注射器吸取5mL RPMI-1640基础培养液,注入小鼠腹腔,轻轻抽回注射器,晃动小鼠腿部和尾部几次。用原注射器抽回腹腔内液体,注入离心管。如此反复操作3~4次。1000r/min离心10min,弃上清。用20~50mL完全培养液重悬细胞,100μL/孔滴加到培养板,置培养箱备用。
(2)脾细胞制备:加强免疫后3d,取免疫Balb/c小鼠,眼眶采血后脱臼处死,在75%酒精中消毒后取脾脏,去除结缔组织,制备脾细胞悬液,转移到50mL离心管中,加RPMI-1640至30mL,1500~2000r/min离心5min,弃上清,加RPMI-1640至30mL,计数待用。
(3)骨髓瘤细胞制备:取3瓶生长状态良好的(活细胞数>95%)骨髓瘤细胞,将之完全吹下,转移到50mL离心管中,加RPMI-1640至30mL,1500~2000r/min离心5min,弃上清,加RPMI-1640至30mL,计数待用。
(4)细胞混合:脾细胞:骨髓瘤细胞=8:1,混合,1500~2000r/min离心5min。
(5)细胞融合:将混合好的细胞离心,倒干上清,把沉淀细胞块弹成糊状,置37℃水浴,在1min内加入1mL融合剂,融合剂为聚乙二醇(PEG)4000,作用2min,并轻轻搅拌细胞,在随后4min内加入20mL无血清的PEG营养液,1000r/min离心10min,弃上清。用20~50mL完全培养液重悬细胞,铺种于含饲养细胞的96孔细胞培养板,每孔100μL,置培养箱中。
3.细胞株筛选
待细胞长至孔底的1/3~1/2时,即可进行抗体检测。采用ELISA方法对有杂交瘤细胞生长的培养孔进行筛选,筛选分两步:第一步先用间接ELISA筛选出阳性细胞孔,第二步选用氟氯氰菊酯为标准品,用间接竞争ELISA对阳性细胞进行抑制效果测定。选出对氟氯氰菊酯标准品具有较好抑制的孔,采用有限稀释法进行亚克隆,用同样的方法进行检测。重复三次,即可得到能稳定分泌氟氯氰菊酯单克隆抗体的细胞株。
4.腹水制备
将液体石蜡注射6~8周Balb/c小鼠,500μL/只。10天后将处于对数生长期的杂交瘤细胞用RPMI-1640基础培养基收集,用血球计数板和显微镜计数,细胞浓度在1.0×106~1.5×106个/mL范围内。每只小鼠0.5mL杂交瘤细胞注射到腹腔。注意观察在一周后小鼠腹部膨大,用无菌注射器于小鼠腹腔采集腹水,每隔一到两天采集一次,这样多次反复采集直到小鼠自然死亡。4℃下5000r/min离心5min,收集上清,并去掉腹水上层漂浮的脂肪和蛋白质膜。
5.抗体纯化
单克隆抗体采用辛酸-硫酸铵方法纯化。
6.抗体效价测定
采用间接ELISA方法测定抗体效价,步骤参考1.中动物免疫的血清效价测定。结果显示,氟氯氰菊酯单克隆抗体的效价≥100000。
7.抗体交叉反应性测定
采用间接竞争ELISA方法测定,结果发现,氟氯氰菊酯单克隆抗体对氟氯氰菊酯及其他菊酯类杀虫剂的交叉反应率为:氟氯氰菊酯为100%,甲氰菊酯、氯氟氰菊酯、氰戊菊酯、氟氰戊菊酯、联苯菊酯、氯菊酯、氯氰菊酯、溴氰菊酯、醚菊酯均<1%。由此可见,所制备的抗体特异性较好。
以上所述实施例仅表达了本发明的实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制,但凡采用等同替换或等效变换的形式所获得的技术方案,均应落在本发明的保护范围之内。
Claims (6)
3.一种氟氯氰菊酯人工抗原,其特征在于,其是载体蛋白和权利要求1所述的氟氯氰菊酯半抗原偶联得到的偶联物。
4.如权利要求3所述的氟氯氰菊酯人工抗原,其特征在于,所述载体蛋白为牛血清白蛋白、鸡卵清白蛋白、人血清白蛋白或血蓝蛋白。
5.一种氟氯氰菊酯抗体,其特征在于,它是由权利要求3所述的氟氯氰菊酯人工抗原经动物免疫得到,其能与氟氯氰菊酯发生特异性免疫反应。
6.一种权利要求5所述的氟氯氰菊酯抗体在检测氟氯氰菊酯残留中的应用。
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